Hemophilia A is a common disorder of blood coagulation due to deficiency of clotting Factor VIII. The gene for factor VIII has been cloned and characterized and a search has begun to understand the molecular basis of hemophilia A. In this proposal, we will characterize all the molecular defects in about 300 patients with various forms of hemophilia A. Mutation detection includes screening of PCR products (by denaturing gradient gel electrophoresis or single stranded electrophoresis or other methods) and nucleotide sequencing of mutant amplified DNA products. Mutations that change amino acids in important areas of the factor VIII protein will be analyzed for their consequences on the function of this protein. The analysis will involve study of factor VIII in CRM positive plasmas and study of factor VIII after transient expression of mutant cDNAs into mammalian cells. The work proposed will provide a clearer understanding of: 1) nature of mutations and prenatal origin and 2) structure-function relationship in the factor VIII protein.